PARK7 maintains the stemness of glioblastoma stem cells by stabilizing epidermal growth factor receptor variant III.

PARK7 is involved in many key cellular processes, including cell proliferation, transcriptional regulation, cellular differentiation, oxidative stress protection, and mitochondrial function maintenance. Deregulation of PARK7 has been implicated in the pathogenesis of various human diseases, including cancer. Here, we aimed to clarify the effect of PARK7 on stemness and radioresistance of glioblastoma stem cells (GSCs). Serum differentiation and magnetic cell sorting of GSCs revealed that PARK7 was preferentially expressed in GSCs rather than differentiated GSCs. Immunohistochemical staining showed enhanced expression of PARK7 in glioma tissues compared to that in normal brain tissues. shRNA-mediated knockdown of PARK7 inhibited the self-renewal activity of GSCs in vitro, as evidenced by the results of neurosphere formation, limiting dilution, and soft-agar clonogenic assays. In addition, PARK7 knockdown suppressed GSC invasion and enhanced GSC sensitivity to ionizing radiation (IR). PARK7 knockdown suppressed expression of GSC signatures including nestin, epidermal growth factor receptor variant III (EGFRvIII), SOX2, NOTCH1, and OCT4. Contrarily, overexpression of PARK7 in CD133- non-GSCs increased self-renewal activities, migration, and IR resistance, and rescued the reduction of GSC factors under shPARK7-transfected and serum-differentiation conditions. Intriguingly, PARK7 acted as a co-chaperone of HSP90 by binding to it, protecting EGFRvIII from proteasomal degradation. Knockdown of PARK7 increased the production of reactive oxygen species, inducing partial apoptosis and enhancing IR sensitivity in GSCs. Finally, PARK7 knockdown increased mouse survival and IR sensitivity in vivo. Based on these data, we propose that PARK7 plays a pivotal role in the maintenance of stemness and therapeutic resistance in GSCs.

Clinical outcomes of primary aldosteronism based on lateralization index and contralateral suppression index after adrenal venous sampling in real-world practice: a retrospective cohort study.

BACKGROUND: We investigated whether the contralateral suppression index in aldosterone-producing adrenal adenoma could be used as a diagnostic criterion when catheterization in either right or left adrenal vein fails or when a discrepancy in the adrenal vein sampling (AVS) results and imaging findings occurs in the real-world practice. METHODS: We retrospectively reviewed 48 patients who had resistant hypertension (HTN) or hypokalemia with a biochemical diagnosis of primary aldosteronism and who underwent AVS from January 2009 to June 2017 at a tertiary referral hospital. Selection index (SI), lateralization index (LI), and contralateral suppression index (CSI) were calculated based on AVS results and the final clinical outcomes were evaluated. RESULTS: The catheterization of both adrenal veins was successful in 43 of 48 (89.6%) patients. The lateralization based only on LI was performed in 23 out of 43 (53.5%) patients. When CSI and LI were combined in decision making, the concordance between adrenal computed tomography scan and AVS for unilateral lesion improved from 59.3% (19/32) to 75.0% (24/32). CSI also correlated well with unilateral adrenal disease in the catheterization failure group. The final outcomes of HTN were better in the contralateral suppression group. CONCLUSION: CSI combined with LI could be a supplementary diagnostic tool in patients with non-lateralization or catheterization failure and predict the clinical outcomes of HTN in patients with primary aldosteronism.

Ultralow Water Permeation Barrier Films of Triad a-SiNx:H/n-SiOxNy/h-SiOx Structure for Organic Light-Emitting Diodes.

Organic electronic devices such as organic light-emitting diodes (OLEDs), quantum dot LEDs, and organic photovoltaics are promising technologies for future electronics. However, achieving long-term stability of organic-based optoelectronic devices has been regarded as a crucial problem to be solved. In this work, a simple and reproducible fabrication method for ultralow water permeation barrier films having a triple-layered (triad) hydrogenated silicon nitride (a-SiNx:H)/nanosilicon oxynitride (n-SiOxNy)/hybrid silicon oxide (h-SiOx) multistructure is presented. Two triad (a-SiNx:H/n-SiOxNy/h-SiOx)n=2 multistructure barrier films are deposited on both sides of a poly(ethylene terephthalate) substrate using a combination of low-pressure plasma-enhanced chemical vapor deposition and dip coating. The deposited films show a high average transmittance (400-700 nm) of 84% and an ultralow water vapor transmission rate of 2 × 10-6 g/m2/day. In the electroluminescence characteristics of OLEDs encapsulated with two triad barrier films, the operational lifetime (T50) of OLEDs is 1584 h, which is almost similar to that (1416 h) of OLEDs encapsulated with a glass lid.

Intestinal Epithelial Deletion of Sphk1 Prevents Colitis-Associated Cancer Development by Inhibition of Epithelial STAT3 Activation.

BACKGROUND AND AIMS: Colitis-associated cancer (CAC) is one of the most serious complications in patients with inflammatory bowel disease. Sphingosine kinase 1 (Sphk1) is a key enzyme in the sphingolipid pathway and has oncogene potential for inducing both initiation and progression of tumors. The aim of this work is to characterize the role of epithelial Sphk1 in mouse colitis and CAC models. METHODS: We investigated the roles of Sphk1 in CAC by conditional deletion of Sphk1 in intestinal epithelial cells (IECs). RESULTS: CAC was induced in both Sphk1ΔIEC/ApcMin/+ and Sphk1IEC/ApcMin/+ mice by administration of 2% dextran sodium sulfate (DSS) for 7 days. Genetic deletion of Sphk1 significantly reduced the number and size of tumors in ApcMin/+ mice. Histologic grade was more severe in Sphk1ΔIEC/ApcMin/+ mice compared with Sphk1IEC/ApcMin/+ mice (invasive carcinoma, 71% versus 13%, p < 0.05). Deletion of Sphk1 decreased mucosal proliferation and inhibited STAT3 activation and genetic expression of cyclin D1 and cMyc in tumor cells. Conditional deletion of Sphk1 using CRISPR-Cas9 in HCT 116 cells inhibited interleukin (IL)-6-mediated STAT3 activation. CONCLUSIONS: Epithelial conditional deletion of Sphk1 inhibits CAC in ApcMin/+-DSS models in mice by inhibiting STAT3 activation and its target signaling pathways.

Early differentiation of long-standing persistent atrial fibrillation using the characteristics of fibrillatory waves in surface ECG multi-leads.

We characterized the f-waves in atrial fibrillation (AF) in the surface ECG by quantifying the amplitude, irregularity, and dominant rate of the f-waves in leads II, aVL, and V1, and investigated whether those parameters of the f-waves could discriminate long-standing persistent AF (LPeAF) from non-LPeAF. A total of 224 AF patients were enrolled: 112 with PAF (87 males), 48 with PeAF (38 males), and 64 with LPeAF (47 males). The f-waves in surface ECG leads V1, aVL, and II, which reflect well electrical activity in the right atrium (RA), the left atrium (LA), and both atria, respectively, were analyzed. The f-waves for LPeAF had lower amplitudes in II and aVL, increased irregularity and a higher dominant rate in II and V1 compared to PAF and PeAF (all p < 0.02). In a multivariate analysis, a low amplitude in lead II (<34.6 uV) and high dominant rate in lead V1 (≧390/min) (p < 0.001) independently discriminated LPeAF from the other AF types. The f-waves combined with both a low amplitude in lead II and high dominant rate in lead V1 were significantly associated with LPeAF (OR 6.27, p < 0.001). Characteristics of the f-waves on the surface ECG could discriminate LPeAF from other types of AF.

PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3.

Microtubule-associated end-binding protein 3 (EB3) accumulates asymmetrically at the tip-end of growing microtubules, providing a central platform for linking various cellular components. EB3 orchestrates microtubule dynamics and targeting, enabling diverse processes within neurons. Inositol 1, 4, 5-trisphosphate 3-kinase A (IP3K-A; also known as ITPKA) is a neuron-enriched protein that binds to microtubules by PKA-dependent manners. In this study, we found that IP3K-A binds to EB3 and their binding affinity is precisely regulated by protein kinase A (PKA)-dependent phosphorylation of IP3K-A at Ser119 (pSer119). We also revealed that the complex of IP3K-A and EB3 dissociates and reassociates rapidly during chemically induced LTP (cLTP) condition. This dynamic rearrangement of IP3K-A and EB3 complex will contribute remodeling of microtubule cytoskeleton allowing effective structural plasticity in response to synaptic stimulations.

Inositol 1,4,5-trisphosphate 3-kinase A overexpressed in mouse forebrain modulates synaptic transmission and mGluR-LTD of CA1 pyramidal neurons.

Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A) regulates the level of the inositol polyphosphates, inositol trisphosphate (IP3) and inositol tetrakisphosphate to modulate cellular signaling and intracellular calcium homeostasis in the central nervous system. IP3K-A binds to F-actin in an activity-dependent manner and accumulates in dendritic spines, where it is involved in the regulation of synaptic plasticity. IP3K-A knockout mice exhibit deficits in some forms of hippocampus-dependent learning and synaptic plasticity, such as long-term potentiation in the dentate gyrus synapses of the hippocampus. In the present study, to further elucidate the role of IP3K-A in the brain, we developed a transgenic (Tg) mouse line in which IP3K-A is conditionally overexpressed approximately 3-fold in the excitatory neurons of forebrain regions, including the hippocampus. The Tg mice showed an increase in both presynaptic release probability of evoked responses, along with bigger synaptic vesicle pools, and miniature excitatory postsynaptic current amplitude, although the spine density or the expression levels of the postsynaptic density-related proteins NR2B, synaptotagmin 1, and PSD-95 were not affected. Hippocampal-dependent learning and memory tasks, including novel object recognition and radial arm maze tasks, were partially impaired in Tg mice. Furthermore, (R,S)-3,5-dihydroxyphenylglycine-induced metabotropic glutamate receptor long-term depression was inhibited in Tg mice and this inhibition was dependent on protein kinase C but not on the IP3 receptor. Long-term potentiation and depression dependent on N-methyl-d-aspartate receptor were marginally affected in Tg mice. In summary, this study shows that overexpressed IP3K-A plays a role in some forms of hippocampus-dependent learning and memory tasks as well as in synaptic transmission and plasticity by regulating both presynaptic and postsynaptic functions.

Reduction of Cav1.3 channels in dorsal hippocampus impairs the development of dentate gyrus newborn neurons and hippocampal-dependent memory tasks.

Cav1.3 has been suggested to mediate hippocampal neurogenesis of adult mice and contribute to hippocampal-dependent learning and memory processes. However, the mechanism of Cav1.3 contribution in these processes is unclear. Here, roles of Cav1.3 of mouse dorsal hippocampus during newborn cell development were examined. We find that knock-out (KO) of Cav1.3 resulted in the reduction of survival of newborn neurons at 28 days old after mitosis. The retroviral eGFP expression showed that both dendritic complexity and the number and length of mossy fiber bouton (MFB) filopodia of newborn neurons at ≥ 14 days old were significantly reduced in KO mice. Both contextual fear conditioning (CFC) and object-location recognition tasks were impaired in recent (1 day) memory test while passive avoidance task was impaired only in remote (≥ 20 days) memory in KO mice. Results using adeno-associated virus (AAV)-mediated Cav1.3 knock-down (KD) or retrovirus-mediated KD in dorsal hippocampal DG area showed that the recent memory of CFC was impaired in both KD mice but the remote memory was impaired only in AAV KD mice, suggesting that Cav1.3 of mature neurons play important roles in both recent and remote CFC memory while Cav1.3 in newborn neurons is selectively involved in the recent CFC memory process. Meanwhile, AAV KD of Cav1.3 in ventral hippocampal area has no effect on the recent CFC memory. In conclusion, the results suggest that Cav1.3 in newborn neurons of dorsal hippocampus is involved in the survival of newborn neurons while mediating developments of dendritic and axonal processes of newborn cells and plays a role in the memory process differentially depending on the stage of maturation and the type of learning task.

The relationship between coronary atherosclerosis and body fat distribution measured using dual energy X-ray absorptiometry.

OBJECTIVE: Body fat distribution is closely related to cardiovascular diseases. We aimed to evaluate the relationship between truncal fat distribution and the extent of coronary atherosclerosis. METHODS: Total body fat and regional body fat distributions were measured using dual-energy X-ray absorptiometry (DXA) in 746 Korean patients who underwent coronary angiography. The ratios of truncal fat mass to total body fat mass (FMtrunk/FMtotal), truncal fat mass to fat mass in both legs (FMtrunk/FMleg) and truncal fat mass to fat mass in both arms (FMtrunk/FMarm) were calculated as representative parameters for truncal fat accumulation. The extent of coronary atherosclerosis was assessed using the Gensini score. RESULTS: The mean Gensini score of the patients was 21.3 ± 24.4. FMtrunk/FMtotal, FMtrunk/FMleg and FMtrunk/FMarm revealed positive correlations with the Gensini score (r = 0.242, p < 0.001; r = 0.219, p < 0.001; r = 0.133, p < 0.001, respectively). In contrast, body mass index (BMI) and total body fat mass did not correlate with the Gensini score. On multiple regression analysis, FMtrunk/FMtotal was associated with the Gensini score independently of age, gender, BMI and major risk factors of coronary heart disease (B = 0.039, p < 0.001). CONCLUSION: Truncal fat distribution is associated with the extent of coronary atherosclerosis and more clinically relevant to that compared with total body fat or BMI in Korean patients.

Fetal hematopoietic stem cells express MFG-E8 during mouse embryogenesis.

The milk fat globule-EGF-factor 8 protein (MFG-E8) has been identified in various tissues, where it has an important role in intercellular interactions, cellular migration, and neovascularization. Previous studies showed that MFG-E8 is expressed in different cell types under normal and pathophysiological conditions, but its expression in hematopoietic stem cells (HSCs) during hematopoiesis has not been reported. In the present study, we investigated MFG-E8 expression in multiple hematopoietic tissues at different stages of mouse embryogenesis. Using immunohistochemistry, we showed that MFG-E8 was specifically expressed in CD34(+) HSCs at all hematopoietic sites, including the yolk sac, aorta-gonad-mesonephros region, placenta and fetal liver, during embryogenesis. Fluorescence-activated cell sorting and polymerase chain reaction analyses demonstrated that CD34(+) cells, purified from the fetal liver, expressed additional HSC markers, c-Kit and Sca-1, and that these CD34(+) cells, but not CD34(-) cells, highly expressed MFG-E8. We also found that MFG-E8 was not expressed in HSCs in adult mouse bone marrow, and that its expression was confined to F4/80(+) macrophages. Together, this study demonstrates, for the first time, that MFG-8 is expressed in fetal HSC populations, and that MFG-E8 may have a role in embryonic hematopoiesis.

PGD2 deficiency exacerbates food antigen-induced mast cell hyperplasia.

Prostaglandin D2 (PGD2) is a major prostanoid secreted mainly by mast cells. Although PGD2 has been identified as a modulator of allergic inflammation, its precise role remains unclear. Here we investigate the role of PGD2 in food allergy. Oral administration of ovalbumin induces allergic responses in sensitized wild-type (WT) mice. Systemic gene deficiency of haematopoietic PGD synthase (H-PGDS(-/-)) exacerbates all of the manifestations accompanying severe mast cell hyperplasia in the intestine. Morphological studies show that c-kit/FcɛRI-positive WT mast cells strongly express H-PGDS. Transplantation of H-PGDS(-/-) mast cells also aggravates ovalbumin-induced mast cell hyperplasia and allergic symptoms in mast cell null mice. H-PGDS deficiency accelerates the production of SDF-1α and the activity of MMP-9 in the antigen-stimulated intestine. SDF-1α receptor blockade or MMP-9 inhibition relieves the exacerbated mast cell hyperplasia and manifestations observed in H-PGDS(-/-). Thus, PGD2 deficiency results in food antigen-induced mast cell hyperplasia.

CCR8 regulates contact hypersensitivity by restricting cutaneous dendritic cell migration to the draining lymph nodes.

Allergic contact dermatitis (ACD) is a typical occupational disease in industrialized countries. Although various cytokines and chemokines are suggested to be involved in the pathogenesis of ACD, the roles of these molecules remain to be elucidated. CC chemokine receptor 8 (CCR8) is one such molecule, of which expression is up-regulated in inflammatory sites of ACD patients. In this study, we found that Ccr8(-/-) mice developed severer contact hypersensitivity (CHS) responses to 2,4-dinitrofluorobenzene, a murine model of ACD, compared with wild-type mice. T cells from Ccr8(-/-) mice showed enhanced proliferative recall responses and Th1 and Th17 cell populations were expanded in these mice. However, CHS responses were similar between SCID mice adoptively transferred with Ccr8(-/-) and wild-type T cells, suggesting that CCR8 in T cells is not responsible for the exacerbation of CHS. Notably, skin-resident dendritic cells (DCs), such as Langerhans cells and dermal DCs, and inflammatory DCs were highly accumulated in lymph nodes (LNs) of Ccr8(-/-) mice after sensitization. Consistent with this, Ccr8(-/-) antigen-presenting cells readily migrated from the skin to the draining LNs after sensitization. These observations suggest that CCR8 negatively regulates migration of cutaneous DCs from the skin to the draining LNs in CHS by keeping these cells in the skin.

Inositol 1,4,5-trisphosphate 3-kinase A is a novel microtubule-associated protein: PKA-dependent phosphoregulation of microtubule binding affinity.

Inositol 1,4,5-trisphosphate 3-kinase A (IP(3)K-A) is a brain specific and F-actin-binding protein. We recently demonstrated that IP(3)K-A modulates a structural reorganization of dendritic spines through F-actin remodeling, which is required for synaptic plasticity and memory formation in brain. However, detailed functions of IP(3)K-A and its regulatory mechanisms involved in the neuronal cytoskeletal dynamics still remain unknown. In the present study, we identified tubulin as a candidate of IP(3)K-A-binding protein through proteomic screening. By various in vitro and in vivo approaches, we demonstrated that IP(3)K-A was a novel microtubule-associated protein (MAP), and the N terminus of IP(3)K-A was a critical region for direct binding to tubulin in dendritic shaft of hippocampal neurons. Moreover, PKA phosphorylated Ser-119 within IP(3)K-A, leading to a significant reduction of microtubule binding affinity. These results suggest that PKA-dependent phosphorylation and microtubule binding of IP(3)K-A are involved in its regulatory mechanism for activity-dependent neuronal events such as local calcium signaling and its synaptic targeting.

CD8 T-cell activation in mice injected with a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean Plasmodium vivax.

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8(+) T-cells and CD4(+) T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8(+) cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.

CXC chemokine receptor 4 expressed in T cells plays an important role in the development of collagen-induced arthritis.

INTRODUCTION: Chemokines and their receptors are potential therapeutic targets in rheumatoid arthritis (RA). Among these, several studies suggested the involvement of CXC chemokine 4 (CXCR4) and its ligand CXC ligand 12 (SDF-1) in RA pathogenesis. However, the role of these molecules in T-cell function is not known completely because of embryonic lethality of Cxcr4- and Cxcl12-deficient mice. In this report, we generated T cell-specific Cxcr4-deficient mice and showed that the CXCR4 in T cells is important for the development of collagen-induced arthritis (CIA). METHODS: T cell-specific Cxcr4-deficient mice were generated by using the Cre-loxP system. Mice harboring loxP sites flanking exon 2 of the Cxcr4gene (Cxcr4flox/flox) were generated by homologous recombination and crossed with Cre transgenic mice expressing Cre recombinase under the control of Lck promoter (Cxcr4+/+/Lck-Cremice) to generate T cell-specific Cxcr4-deficient mice (Cxcr4flox/flox/Lck-Cre mice). CIA was induced by immunization with chicken type II collagen and Complete Freund's Adjuvant (CFA). RESULTS: The incidence, but not the severity, of CIA was significantly reduced in Cxcr4flox/flox/Lck-Cre mice compared with Cxcr4+/+/Lck-Cre mice. We found that the expression of CXCR4 was enhanced in activated T cells, and the migration of Cxcr4-deficient T cells toward SDF-1 was severely impaired. However, antibody production, cellular proliferative response, and cytokine production on treatment with type II collagen (IIC) were normal in these knockout mice, suggesting that CXCR4 is not involved in T-helper functions. Interestingly, the proportion of CXCR4-expressing T cells was much increased in affected joints compared with that in draining lymph nodes in CIA-induced mice, and distribution of Cxcr4flox/flox/Lck-Cre mouse-derived T cells into affected joints was suppressed compared with that in Cxcr4+/+/Lck-Cre T cells. CONCLUSIONS: These results indicate that CXCR4 expression in T cells is important for the development of CIA, by recruiting activated T cells toward inflammatory sites, and suggest that CXCR4 is a good target for the treatment of RA in humans.

The mechanism of LPS-induced HIV type I activation in transgenic mouse macrophages.

In the course of the development of acquired immunodeficiency syndrome (AIDS), bacterial infection causes deleterious effects on the progression of the disease; bacterial LPS in the circulation activate immune cells, resulting in the acceleration of HIV replication. However, the precise HIV activation mechanisms in infected hosts remain largely unknown. Previously, we generated transgenic (Tg) mice carrying the HIV type I (HIV-1) genome and showed that LPS induces the activation of HIV-1 in splenocytes through the induction of tumor necrosis factor (TNF) and IL-1, although similarly induced IFN-gamma and IL-6 are not involved. In this study, we analyzed the mechanisms of HIV-1 activation in macrophages using these HIV-1 Tg mice, because macrophages are one of the major reservoirs of HIV-1. In contrast to splenocytes, direct Toll-like receptor (TLR) 4 signaling rather than TLR-induced pro-inflammatory cytokines was responsible for the LPS-induced activation of HIV-1 in macrophages, because the time course of HIV-1 activation was earlier than that observed in splenocytes and TNF neutralization did not inhibit the activation. p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB) activation, but neither extracellular signal-regulated kinase nor c-Jun N-terminal kinase activation, were required for the activation, because only inhibitors for p38 MAPK and NF-kappaB suppressed activation of HIV-1. Furthermore, we showed that myeloid differentiation primary response gene (MyD) 88, rather than Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF), was required as an adaptor molecule for this activation using Myd88(-/-) mice and Dynasore, a specific inhibitor for TRIF, and small interfering RNAs specific for Myd88 and Trif. These observations suggest that suppression of these molecules, which are involved in the TLR4-MyD88 pathway and the downstream p38 MAPK and NF-kappaB pathways, should be beneficial to prevent development of AIDS in HIV-1-infected people.

Regulation of AHI1 expression in adult rat brain: Implication in hypothalamic feeding control.

Recent studies revealed that Abelson helper integration site 1 (AHI1) plays a role in brain development. However, little is known about the role of AHI1 in adult brain. To directly assess the role of AHI1 in the adult brain, we cloned full-length cDNA of rat AHI1 and observed prominent expression of AHI1 in the hypothalamus, which contributes mainly to the control of energy homeostasis. Furthermore, we demonstrated that food deprivation caused induction of AHI1 in the hypothalamus and subsequent re-feeding down-regulated AHI1 expression, suggesting the involvement of AHI1 in feeding control. Moreover, the expression of AHI1 was increased in serum-depleted Neuro2A cells and restored by subsequent insulin treatment. Furthermore, treatment in food-deprived rat with intraperitoneal glucose also reduced the increased AHI1 expression. These results demonstrate that AHI1 expression can be regulated through diet and suggest the novel role of AHI1 in feeding behavior.

CXC chemokine ligand 12 promotes CCR7-dependent naive T cell trafficking to lymph nodes and Peyer's patches.

A number of chemokines, including CCL21, CCL19, CXCL12, and CXCL13, are coexpressed on the lumen or basal lamina of high endothelial venules (HEVs) in lymph nodes (LNs) and Peyer's patches (PPs), consistent with the idea that they might cooperate to regulate lymphocyte trafficking into these lymphoid tissues. In this study we report that CXCL12, acting through its receptor, CXCR4, cooperates with CCR7 ligands to promote T cell trafficking across HEVs. CXCL12 enhanced the CCR7-induced chemotaxis of wild-type but not CXCR4-deficient T cells in vitro at suboptimal concentrations of a CCR7 ligand, but without affecting the expression level or ligand-binding ability of CCR7. Real-time chemotaxis analysis showed that CXCL12 substantially shortened the lag time before cell migration began in vitro, but not the migration speed of T cells responding to suboptimal CCR7 ligand concentrations. In addition, CXCL12 augmented the CCR7 ligand-driven ERK phosphorylation and actin polymerization in T cells under the same conditions. In adoptive transfer experiments, CXCL12 promoted naive T cell trafficking to LNs and PPs in wild-type but not CCR7 ligand-deficient plt/plt recipient mice; this increased T cell trafficking was associated with enhanced binding of the T cells to HEVs and their subsequent migration into the LN parenchyma. Thus, CXCL12 synergizes with CCR7 ligands to promote T cell migration by sensitizing T cells through CXCR4, thus enabling them to respond to lower concentrations of CCR7 ligands. Such concerted action of chemokines provides an additional, previously unknown mechanism for efficient lymphocyte trafficking across HEVs into LNs and PPs.

Nuclear import of the preintegration complex is blocked upon infection by human immunodeficiency virus type 1 in mouse cells.

Mouse cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of host range barriers at steps including virus entry, transcription, RNA splicing, polyprotein processing, assembly, and release. The exact mechanisms for the suppression, however, are not completely understood. To elucidate further the barriers against HIV-1 replication in mouse cells, we analyzed the replication of the virus in lymphocytes from human CD4/CXCR4 transgenic mice. Although primary splenocytes and thymocytes allowed the entry and reverse transcription of HIV-1, the integration efficiency of the viral DNA was greatly reduced in these cells relative to human peripheral blood mononuclear cells, suggesting an additional block(s) before or at the point of host chromosome integration of the viral DNA. Preintegration processes were further analyzed using HIV-1 pseudotyped viruses. The reverse transcription step of HIV-1 pseudotyped with the envelope of murine leukemia virus or vesicular stomatitis virus glycoprotein was efficiently supported in both human and mouse cells, but nuclear import of the preintegration complex (PIC) of HIV-1 was blocked in mouse cells. We found that green fluorescent protein (GFP)-labeled HIV-1 integrase, which is known to be important in the nuclear localization of the PIC, could not be imported into the nucleus of mouse cells, in contrast to human cells. On the other hand, GFP-Vpr localized exclusively to the nuclei of both mouse and human cells. These observations suggest that, due to the dysfunction of integrase, the nuclear localization of PIC is suppressed in mouse cells.